1. Field of the Invention
The present invention relates to a process for hydrolysis of protein which is conducted prior to amino acid hydrolysis. More particularly, the invention provides a new process for hydrolyzing protein adsorbed on a solid surface by a solid phase/gas phase reaction with a vaporized acid mixture.
2. Description of the Prior Art
The hydrolysis of protein which is a pretreatment to be conducted prior to amino acid analysis has been carried out with distilled, azeotropic hydrochloric acid at 100.degree. to 110.degree. C. in an air-free sealed tube for a time of as long as 24 to 144 hours. It is a major disadvantage of the conventional process to heat starting materials for such a long period. While, according to a method of Tsugita et al. developed recently, the hydrolysis can be carried out rapidly in 25 to 50 min with a mixed solution of hydrochloric acid (HCl), trifluoroacetic acid (CF.sub.3 COOH; hereinafter referred to as TFA) and water (H.sub.2 O) [see references (1) to (3)]. The gist of the process developed by Tsugita et al. is as follows:
(i) A high reaction temperature is employed so as to enhance the hydrolysis reaction rate. When the temperature is elevated by 10.degree. C., the reaction rate is approximately doubled. When the temperature is elevated by 60.degree. C., the reaction rate is increased by 64-fold (=2.sup.6). Namely, the unit "hour" may be replaced roughly by "minute" in this case.
(ii) When an organic solvent, particularly a strongly acidic organic acid, is added to protein, a hydrophobic peptide moiety can be hydrolyzed easily.
The fact that the hydrolysis rate is increased by the temperature rise will now be described. A relationship between the hydrolysis rate of Val-Glu dipeptide which is considered to be difficultly hydrolyzable with an acid and the temperature is shown in FIG. 5. The hydrolysis time was 25 min. For comparison, the results obtained by using 6M-HCl are also shown. It is apparent from FIG. 5 that the reaction rate is increased as the temperature is elevated. The reaction rate obtained when a mixture of TFA and hydrochloric acid [CF.sub.3 CO.sub.2 H:HCl (1:2)] was used was far higher than that obtained when 6M-HCl was used.
The effects of the organic acids added to protein will be shown below. The results obtained by using volatile organic acids having relatively excellent effects are summarized in Table 1. It is apparent from this Table that the effect obtained by the addition of TFA is most excellent.
TABLE 1 ______________________________________ Recovery of amino acids from Val--Glu ______________________________________ formic acid:HCl 1:1 85% 1:2 95% acetic acid:HCl 1:1 97% 1:2 100% TFA:HCl 2:1 85% 1:1 100% 1:2 100% propionic acid:HCl 1:1 90% 1:2 97% ______________________________________
It will be understood from the above-described facts that the elevation of the hydrolysis temperature and the addition of TFA are most effective in accelerating the hydrolysis.
In an experiment, myoglobin, one of known proteins, was hydrolyzed with TFA/hydrochloric acid (1:2) for 10 and 25 minutes and the decomposition rate of the protein was determined from the recovery of alanine to obtain the results shown in FIG. 6. It is apparent that 100% recovery was obtained at around 166.degree. C. after 25 minutes.
Though the hydrolysis process developed by Tsugita et al. wherein a mixed solution of TFA and water is used is capable of completing the hydrolysis rapidly within 20 to 50 minutes, it has a defect that the formed amino acids are contaminated with a solvent used in this solution process to cause an error in the analysis valued in terms the amino acids. Therefore, a complicated evaporation procedure is necessitated in this process for removing the acid after the hydrolysis to make the automatic operation thereof difficult. In addition, a long time is required prior to the analysis of the amino acids and, therefore, synchronization with the amino acid analysis device is difficult.